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Effects of varying concentrations of Morinda lucida leaf extract on in vitro gas production and fermentation parameters

T J Adebayo, K O Adebayo, R Y Aderinboye and O S Sowande

Federal University of Agriculture, Abeokuta, Nigeria


Morinda lucida is an evergreen, multipurpose, medium-sized tree that is well known for its astringent bitter taste and used in the treatment of many diseases in many parts of the tropics. Previous reports have shown that it contains secondary compounds that can aid in rumen modification and reduce methanogenesis. This study was designed to evaluate the effects of aqueous Morinda lucida leaf extract (MLE) on gas production and fermentation parameters in an in vitro medium. The leaf powder (0g, 2g, 4g, 6g, 8g, and 10g) was extracted by maceration in 100 ml distilled water for 3 hours with intermittent agitation to give 0, 2, 4, 6, 8 and 10% concentrations (which served as the treatment groups) respectively. Incubation was carried out for 48 hours at 390C (±1) using 100 ml calibrated glass syringes, filled with 200 mg of Panicum maximum and concentrate in a ratio of 60:40, 30 ml of the inoculum, and 2 ml of varying concentrations of MLE. Total gas volume, methane production, dry matter digestibility, short-chain fatty acids, and metabolizable energy were estimated after 48 h incubation period. The phytochemical composition of MLE was determined. The experiment was arranged in a completely randomized design (CRD).

Morinda lucida leaf extract contains high flavonoid, phenolic acid, and tannin along with other phytochemicals. It depressed (p < 0.05) methane gas production without any negative effect on total volatile fatty acid production up to 10% concentration. The highest methane depression (19%) was obtained at 10% concentration. Thus, MLE can be used to manipulate rumen fermentation at 10% concentration.

Keywords: digestibility, methanogenesis, phytogenic, rumen modification


In vitro study appears to be a prerequisite to assessing the suitability of feed ingredients and feed additives that can mediate methane production. It has the advantage of being less expensive and minimizing experimental animal usage in research (Yáñez-Ruiz et al 2016). Positive results have been reported on methane gas reduction potentials of phytogenic plants during in vitro studies (Dong et al 2010; Aderinboye et al 2020; Adetunji et al 2020). The reports of the authors have suggested that the manipulation potential of the phytogenic plants is deeply rooted in the type, number, and quantity of secondary metabolites they contain.

Methane (CH4) is one of the greenhouse gases produced during the fermentation of organic matter in the rumen. It has a global warming potential of 25-fold more than carbon dioxide (CO2) (Broucek 2014), a longer atmospheric lifetime (Thorpe 2009), and about 12 % dietary energy loss (Johnson and Johnson 1995), thereby posing a great threat to environmental sustainability and animal productivity (Aderinboye et al 2020). Enterically produced methane from ruminant animals is an inevitable natural process required to prevent hydrogen accumulation produced by the microbial population in the rumen during feed digestion (Martin et al 2010). Methanogenesis in ruminant production can thus not be eradicated but could be minimized through various rumen manipulation strategies (Bodas et al 2009; Adebayo et al 2017; Adetunji et al 2020). Carefully designed dietary management and rumen modification strategies tagged as the ‘preventative measure’ and ‘end of pipe measure’ respectively by (Clemens and Ahlgrimm 2001) have been adopted to reduce methanogenesis in ruminant husbandry. The former involves the use of non-protein-Nitrogen (NPN) such as urea in feeding and supplementing rumen degradable carbohydrates (high grain diets) (Wanapat 2000) while the latter involves the inclusion of additives in the form of antibiotics, defaunation agents, probiotics and phytogenic (McAllister and Newbold 2008) that will change the rumen environment. The residual effect of synthetic chemical substances used in modifying ruminal fermentation (García-González et al 2006), has resulted in restrictions being placed on their sub-therapeutic usage in animal production. Recent studies (Choubey et al 2014; Adebayo et al 2019; Aderinboye et al 2020) have shown that many tropical plants have more benefits (as rumen manipulators) beyond their nutrient content (Teferedegne 2000) and could be used in place of the synthetic chemicals.

Morinda lucida, a tropical multi-purpose tree, belongs to the plant family, Rubiaceae and grows widely in many tropical countries where it is used in the treatment of various diseases such as malaria, infertility, and diabetes (Raji et al 2005; Olayemi et al 2016; Osuntokun et al 2016; Igwilo et al 2018). Ugbeni and Osubor (2019) reported the presence of phytochemicals like saponin, alkaloid, flavonoid, tannin, phytate, oxalate, anthraquinones, and cyanogenic glycosides in the plant. Phytochemicals in many tropical plants have varied pharmacological actions in the body including disease prevention (Fajimi and Taiwo 2005); feed digestion, metabolism, and nutrient utilization resulting in better performance of the animals (Windisch et al 2008). For ruminants, it influences the digestion processes in terms of site of digestion (Teferedegne 2000; Preston et al 2021), the rate and products of fermentation through rumen modification. More importantly to ruminant nutritionists is the phytogenic effects on rumination gases such as ammonia and methane that accelerate global warming and reduce animal productivity. The nutritive value of the plant in ruminant feeding was reported by Osakwe and Drochner (2006). This research work was, however, designed to assay the potentials of varying concentrations of aqueous extract of Morinda lucida leaves on in vitro gas production, fermentation, and post-incubation parameters.

Materials and methods

Experimental site

The experiment was carried out at the Laboratory of Animal Nutrition Department, Federal University of Agriculture, Abeokuta (FUNAAB), Ogun State, Nigeria.

Experimental materials and preparation

Morinda lucida plants were harvested around FUNAAB, the leaves were detached, washed, and air-dried under shade to constant weight before milling. Various quantities of the leaf powder (2g, 4g, 6g, 8g, and 10g) were extracted by maceration in 100 ml distilled water for 3 hours with intermittent agitation (Nworu et al 2012). The solution was filtered using Whatman no1 filter paper and kept in the refrigerator at 40C. The experimental substrates were Panicum maximum and concentrate. Concentrate supplement was formulated to contain 14% crude protein deemed adequate for growing goats (NRC 1981). The concentrate’s composition was wheat offal (50%), maize bran (30%), palm kernel cake (17%), bone meal (2%), and salt (1%).

Quantitative analysis of phytochemicals in Morinda lucida leaf extract

The phytochemicals in MLE were determined thus: Tannin (Singleton and Rossi 1965; Velioglu et al 1998; Marinova et al 2005), Alkaloid (Harborne 1973), Phenolic acid (Singleton et al 1999), Flavonoid using quantification procedure (Amorim et al. 2008), Cyanogenic Glycosides (Riales and Albrink 1981; Drochioiu et al 2008), Oxalate by titration procedure (Mishra et al 2017), Saponin (Harborne 1973; Obadoni and Ochuko 2002), Phytic acid by the iron titration procedure (Reeves 1979) and calculation formula (Garcia-Villanova and de 1982) and Trypsin inhibitor (Hamerstrand et al 1981).

Experimental design and procedure

The experiment was arranged in a completely randomized design (CRD) with 6 treatments (0, 2, 4, 6, 8, and 10% concentrations of MLE) replicated 9 times. Incubation was carried out for 48 hours at 390C (±1) as described by (Menke and Steingass 1988) under continuous flushing with CO2. The rumen content of a bull was collected in a pre-warmed flask immediately after slaughtering in the abattoir and taken immediately to the laboratory and filtered before mixing with buffer solution (NaHCO3+3Na2HPO4+KCl+NaCl+MgSO4.7H 2O+CaCl2.2H2O) in ratio 1:2 to make the inoculum. Hundred (100) ml calibrated glass syringes were filled with 200 mg of Panicum maximum and concentrate in the ratio of 60:40, 30 ml of the inoculum, and 2 ml of varying concentrations of MLE. The silicon tube in the syringes was tightened by a metal clip to prevent the escape of gas during incubation. Three blanks containing 40 ml of medium only was included in each run to correct for gas production outside the substrate fermentation. The volume of gas produced was measured at three hours intervals, 0 to 48 hours. During the post-incubation period, total gas volume was measured; net gas volume was determined by subtracting the blank value from total gas volume; methane production was determined from the gas production at the 48th hour by introducing 4 ml of NaOH (10M) as reported by (Fievez et al 2005).

Estimation of post-incubation parameters

The post incubation parameters were estimated at 24h post gas collection from 3 syringes per treatment as follows: Metabolizable energy (ME) = (Menke and Steingass 1988), Organic matter digestibility (OMD) (%) = (Menke and Steingass 1988) and short-chain fatty acids (SCFA) = (Getachew et al 2002).

In vitro dry matter digestibility

Residues from three syringes were decanted into pre-weighed crucibles after 48 hours of degradation. These were oven-dried at 1050C for 24 hours, then weighed. Degradability was determined as the difference between the quantity of undegraded residue and quantity of substrate incubated, then related their difference as a percentage of the substrate incubated.

Rumen fermentation parameters

After 48 hours of incubation, 5 ml of the incubated fluid was transferred from three syringes per treatment into plastic bottles to which 1 ml of metaphosphoric acid was added to stop microbial fermentation and left for 30 minutes before centrifuged at 2500 rpm for 10 min. This portion was used to determine total volatile fatty acid (TVFA) according to (Barnett and Reid 1956). About 10 ml of the supernatant (rumen fluid) was collected and titrated with 0.1N NaOH using phenolphthalein as an indicator to analyze NH 3-N as described by (Ogubai and Sereke 1997).

Statistical Analysis

All data collected were subjected to one-way analysis of variance (ANOVA) in a Completely Randomized Design (CRD) using SAS (2001) procedure, means that were significant were separated using Duncan Multiple Range Test at 5% level of significance.


The phytochemical composition of Morinda lucida leaf extract presented in Table 1 revealed the presence of tannins, phenolic acid, phytate, oxalate, glycoside, flavonoid, and trypsin inhibitor, saponin, and alkaloids.

Table 1. Phytochemical composition of Morinda lucida leaf extract


Morinda lucida leaf extract
composition (mg/100ml)



Phenolic acid






Cyanogenic Glycoside




Trypsin inhibitor


Saponin (%)


Alkaloids (%)


The effect of MLE on in vitro gas production at the 48th hour is presented in Figure 1. The reduction in the volume of gas produced between the control and treatment groups was not noticeably different.

Methane production and percentage methane production is shown in Figure 2 and 3 respectively. Methane production reduced in substrates containing MLE, with 8 % and 10 % concentrations having the least value.

Figure 1. Effect of varying concentrations of Morinda lucida leaf extract
on gas production at 48th hour incubation period

Methane percentage in the gas produced also follows the same trend. Percentage methane depression compared with the control was between 10 % to 19 %.

Figure 2. Effect of varying concentrations of Morinda lucida
leaf extract on methane production
Figure 3. Percentage methane production (%) of substrates containing
varying concentrations of Morinda lucida leaf extract

Morinda lucida leaf extract reduced (p<0.05) IVDMD (Figure 4 and Table 3) with the least value of 50.00 % from the substrate containing 6, 8, and 10 % MLE and the highest value (63.33 %) from substrate containing 0 % MLE.

Figure 4. Effect of varying concentrations of Morinda lucida leaf extract on digestibility

Ammonia Nitrogen (Figure 5 and Table 2) was influenced (p<0.05) by the addition of MLE. The lowest value (39.97 %) was recorded in 6 % MLE while the highest value (54.43 %) 0 % MLE.

Figure 5. effect of varying concentrations of Morinda lucida leaf extract on Ammonia Nitrogen

Post rumen fermentation analysis in Table 2 revealed no differences in pH and total volatile fatty acids of the substrates regardless of the concentration of MLE used. Metabolizable energy (ME) values ranged from 6.85 MJ/KgDM in substrate containing 0 % MLE to 7.44 MJ/KgDM, in substrate containing 10 % MLE. Organic matter digestibility values ranged from 51.90 % in substrate containing 10 % MLE to 55.75 % in substrate containing 0 % MLE. The substrate with 0 % MLE recorded the least value (0.61 mmol) while the highest value (0.71 mmol) was recorded for the substrate with 10 % MLE for SCFAs.

Table 2. Fermentation parameters and microbial count analyses of the substrate containing varying concentrations of Morinda lucida leaf extract




























NH3-N (mg/dl)









ME (MJ/kgDM)









OMD (%)









SCFA (µmol)


















a,b Means in the same row with different superscripts are significantly different (p < 0.05). ME= Metabolizable energy, OMD= Organic Matter Digestibility, SCFA= Short Chain Fatty acids, IVDMD= In vitro dry matter digestibility, TVFA= Total volatile fatty acids, NH3-N= Ammonia Nitrogen, TBC=total bacteria count, TFC= total fungi count, TPC= total protozoan count


Previous studies (Ogundare and Onifade 2009; Nworu et al 2012; Osuntokun et al 2016; Igwilo et al 2018; Ugbeni and Osubor 2019) reported that Morinda lucida contains several biologically active constituents such as tannin, saponin, phenolic acid, phytate, oxalate, glycoside, and flavonoid including terpenoids and resins (Unekwuojo 2011; Appiah-Opong et al 2016) in every part of the plant (stem, leaves, and fruits). Using the same processing method, tannin reported in L. leucocephala (0.015) and M. indica (0.011) by Adebayo et al (2019) is similar to the value recorded in the present study for tannin. However, the authors reported a higher saponin value (6.67 and 3.77 for L. leucocephala and M. indica, respectively) than found in this study. The presence of phytochemicals in Morinda lucida most essentially tannin and saponin suggests that it has methane mitigation potential for ruminant animals (Patra et al 2006; Bodas et al 2009).

The pattern of gas production observed in this study indicates that MLE up to 10% concentration did not have a notable effect on the amount of gas produced by the microbes. The result of this study contradicts the result of Choubey et al (2014) who reported that in vitro supplementation of composite phytogenic feed additives (cPFA) reduced total gas production but in line with Bodas et al (2009) who reported that none of the six phytogenic plants used (Carduus pycnocephalus, Populus tremula, Prunus avium, Quercus robur, Rheum nobile and Salix caprea) affected in vitro gas production. Reduction in methane production from ruminants is expected to improve the animals’ performance and reduce GHG concentration in the atmosphere (Adebayo et al 2021) as more energy will be retained in the animals’ bodies rather than be eructed to the atmosphere. Several studies (Bodas et al 2009; Dong et al 2010; Choubey et al 2014; Adebayo et al 2021) had reported reductions in in vitro methane production with different phytogenic plants. The reduction observed in groups containing MLE compare to control may be because of tannin and saponin present in the extract, since the metabolites have been proved to have the potential for enhancing the flow of microbial protein from the rumen, increasing efficiency of feed utilization, and decreasing methanogenesis (Woodward et al 2001; Goel and Makkar 2012; Preston et al 2021).

The extent of gas production reflects the rate of degradation in feed (Fievez et al 2005; Aderinboye et al (2016), however, lower dry matter digestibility (DMD) was found with increasing concentration of MLE. In line with this report, Bodas et al (2009) reported a methane reduction and enhanced DMD with no effect on gas production. This opposes the report of Kim et al (2019) who found a significantly higher IVDMD when supplementing with essential oil mixtures. Higher in vitro dry matter digestibility observed at 0% concentration of MLE could be responsible for slightly higher gas production observed in the group. Ammonia-nitrogen was reduced as MLE concentration increased. Ammonia nitrogen is an essential source of nitrogen for microbial protein synthesis (Wanapat 2000). This suggests that, at a higher concentration of MLE, the secondary metabolites bind to proteins to hinder its breakdown into ammonia-nitrogen by microbes. Tannins protect proteins from rumen degradation so they can escape into the distal end of the digestive tract where they are more efficiently digested and absorbed (Preston et al 2021). Adetunji et al (2020) also reported a significant reduction in Ammonia-N with cashew nutshell liquid (CNSL) inclusion.

Fermentation parameters could be determined from the gas produced by degraded substrates (Babayemi et al 2009). Just as gas production in the inoculum was not notably affected by varying concentrations of MLE, metabolizable energy (ME), organic matter digestibility (OMD), and short-chain fatty acids (SCFA) were also not influenced by the addition of MLE. However, Adetunji et al (2020) reported a significant reduction in short-chain fatty acid, metabolizable energy, and organic matter degradability with increasing cashew nutshell liquid (CNSL) inclusion levels. The disparity in the results could be due to different test ingredients, techniques, and varying concentrations of metabolites in the test ingredients used. There were no differences in the total volatile fatty acids (TVFAs) concentration produced across the treatments. The energy requirement of ruminant animals is mainly supplied by VFAs. The pH range was not significantly different in this study. Similar values (6.33 – 6.44) on pH were given by Adebayo et al (2017) with no significant difference. This range of pH is considered appropriate for microbial function because microbial yield will reduce greatly when pH approached 5.7 to 6.15 (Pitt et al 1996). Phytogenic products are good rumen modifiers (Dong et al 2010), total phenols and tannins from these products can help to predict their methane mitigation potential (Goel and Makkar 2012). Ten percent (10 %) concentrations of MLE gave the highest methane depression and NH3-N production and digestibility were also lower at this concentration, it is therefore recommended to be used for in vivo experiments to ascertain the finding of this study.



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